The goal of this project is to elucidate structure-function relationships in macromolecular machines. During FY12, we studied: chaperone-assisted proteases involved in protein quality control and cell regulation; membrane remodeling; and two components of pathogenic bacteria. (1) All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. These activities are carried out by energy-dependent proteolytic machines, which consist of two subcomplexes - a protease and an ATPase/unfoldase. Since 1995, we have studied the Clp complexes of E. coli, considered as a model system. We showed that the protease ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpPs. We went on to show that substrate proteins bind to distal sites on the ATPase and are then unfolded and translocated axially into the digestion chamber of ClpP. These studies all dealt with homomeric ring complexes. ClpP in plants depart from this paradigm in being heteromeric. Chloroplast ClpPs associate multiple different subunits. In FY13, we completed and published our structural analysis of ClpP from the green alga Chlamydomonas reinhardtii (1). This ClpP has both active subunits (3 ClpPs) and inactive subunits (5 ClpRs). It also has two ClpT subunits which show Clp-N domains. Negative staining electron microscopy showed that the Chlamydomonas ClpP complex retains the barrel-like shape of homomeric ClpPs, with 4 additional peripheral masses which may represent either the additional IS1 domain of ClpP1 (a feature unique to algae), or ClpTs, or extensions of ClpR subunits. (2) Membrane Remodeling. Remodeling, a process in which lipid bilayer structures are reconfigured by interacting proteins, is central to the functioning and metabolism of cells. We are investigating this phenomenon by cryo-electron microscopy (EM) and cryo-electron tomography (ET) applied to several systems. In FY13, our main efforts were directed towards: (2a) characterizing the effects of the protein alpha-synuclein (aS) on lipid vesicles; and (2b) investigating the polymorphism of clathrin-coated vesicles (2a) aS, which is natively unfolded in solution, accumulates as amyloid fibrils in neurological tissue in patients afflicted with Parkinsons disease, and also interacts with membranes under both physiological and pathological conditions. We used cryo-EM in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the structures produced by adding aS to (initially spherical) lipid vesicles (6). The products obtained depend on the protein: lipid ratio. At a molar ratio of < 1: 40, POPG vesicles are converted into cylindrical micelles 5nm in diameter, together with bilayer tubes of various widths (15 - 50 nm). Between 1 : 20 and 1 : 10, cylindrical micelles are produced exclusively. Other negatively charged lipids (DMPG, DLPG, DAPG) exhibit generally similar behavior. At higher protein: lipid ratios (and RT; 1 : 10), cylindrical micelles are replaced by nanoparticles, whose shape (ellipsoidal) and dimensions (in the 7-10 nm range) resemble those previously observed to be formed by apolipoproteins in the presence of vesicles. Similar nanoparticles formed when aS was added to vesicles of mitochondrial lipids. These observations suggested a mechanism for the previously reported disruption of mitochondrial membranes by aS. Circular dichroism and 4-pulse DEER experiments reveal that in nanoparticles aS assumes a broken helical conformation distinct from the extended helical conformation adopted when aS is bound to intact vesicles or membrane tubules. We also observed aS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that aS can interact with fatty acids and lipids in similar manners. aS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein (8). 2b) Clathrin coats serve as dynamic scaffolds that stabilize membrane curvature during endocytosis and vesicular trafficking. These structures are highly polymorphic. To better appreciate the clathrin lattices and their interactions with vesicles and adaptor proteins, we used cryo-ET to visualize coated particles isolated from bovine brain (3). They range from 66 to 134 nm in diameter. Only particles larger than 80 nm - 20% of the total - contain vesicles. The remaining 80% are clathrin baskets, artifactual assembly products. In CVs, the interstitial space between the coat and the vesicle is occupied by numerous densities, putatively adaptor proteins. Many of the vesicles are offset to one side within the coat, leaving a crescent of interstitial space for adaptor proteins and other components. Several densities - presumably cargo proteins - are associated with the vesicle interior, with only a few in any given vesicle. The combined masses of clathrin coat, adaptor proteins, and vesicle membrane constitute almost all of the mass of a CV, leaving little for cargo. The assembly of a CV in endocytosis therefore represents a massive biosynthetic effort to internalize a relatively diminutive payload, apparently a rather inefficient process. This investment may be needed to overcome the resistance to forming highly curved membranes during endocytosis. 3) The resistance of pathogenic bacteria to conventional antibiotic drugs is a growing problem in combating human infections. An alternative therapeutic strategy uses virus-derived proteins, called lysins, to kill Gram-positive bacteria by damaging the peptidoglycan layer in their envelopes. However, a major obstacle to this approach is that lysins cannot cross the protective outer membrane. To address it, a chimeric lysin was produced and shown to kill Gram-negative bacteria. A crystal structure was obtained for a Yersinia pestis toxin called pesticin and used to guide the design of a hybrid lysin in which a domain from pesticin was fused withT4 lysozyme, an archetypal lysin. This hybrid protein was shown to cross the outer membrane and kill E. coli cells as well as Yersinia strains. Cryo-EM of whole bacterial cells treated with the lysin showed extensive disruption of their membranes (7). Importantly, killing only affects cells that produce the membrane protein FyuA4, a virulence factor, and can therefore be used against essentially any pathogenic strain that expresses FyuA4. 4) Neisseria are Gram-negative bacteria of which two pathogenic species cause gonorrhea, meningitis, and other systemic infections. Neisseria require iron for survival and virulence. While most Gram-negative bacteria secrete siderophores to scavenge iron from the environment, Neisseria extracts iron directly from human sources such as transferrin (Tf). The Neisserial Tf transport system consists of two large surface proteins: Tf-binding protein A (TbpA), a 100 kDa integral outer membrane protein, and TbpB, an 80 kDa Tf receptor. TbpA binds apo and holo transferrin, whereas TbpB associates only with the iron-bound transferrin (Tf). TbpA and TbpB induce bactericidal antibodies in mice, making both proteins important vaccine targets. To elucidate how they function to selectively bind Tf and extract itsbound iron, we combined X-ray crystallography, small angle X-ray scattering, and EM to determine the structure of the 260-kDa iron import complex (7). our The results explain the specificity of TbpA for human Tf, show how TbpA promotes iron release from Tf, and elucidate how TbpB facilitates this process.